High density arrays formed by spatially addressable synthesis of bioaffinity tags on a 2-dimensional solid support have greatly enhanced and simplified the process of biological research and development, since they were invented in the early 1990s. See Science, 251, 767-773, 1991. The key to current microarray technology is deposition of a bioactive agent at a single spot on a microchip in a “spatially addressable” manner.
Current technologies have used various approaches to fabricate microarrays. For example, U.S. Pat. Nos. 5,412,087, and 5,489,678 demonstrate the use of a photolithographic process for making peptide and deoxyribonucleic acid (DNA) microarrays. The patents teach the use of photolabile protecting groups to prepare peptide and DNA microarrays through successive cycles of deprotecting a defined spot on a 1 cm×1 cm chip by photolithography, then flooding the entire surface with an activated amino acid or DNA base. Repetition of this process allows construction of a peptide or DNA microarray with thousands of arbitrarily different peptides or oligonucleotide sequences at different spots on the array. This method is expensive.
An ink jet approach is being used by others, for example, in U.S. Pat. Nos. 6,079,283, 6,083,762, and 6,094,966, to fabricate spatially addressable arrays, but this technique also suffers from high manufacturing cost in addition to the relatively large spot size of 40 to 100 μm. Because the number of bioaffinity tags to be placed on a single chip usually runs anywhere from 1000 to 100000 probes, the spatial addressing method is intrinsically expensive regardless of how the chip is manufactured.
An alternative approach to the spatially addressable method is the concept of using fluorescent dye incorporated polymeric microspheres to produce biological multiplexed arrays. U.S. Pat. No. 5,981,180 discloses a method of using color coded microspheres in conjunction with flow cytometry to perform multiplexed biological assay. Microspheres conjugated with DNA or monoclonal antibody probes on their surfaces were dyed internally with various ratios of two distinct fluorescence dyes. Hundreds of “spectrally addressed” microspheres were allowed to react with a biological sample and the “liquid array” was analyzed by passing a single microsphere through a flow cytometry cell to decode sample information.
U.S. Pat. Nos. 6,023,540 and 6,266,459 disclose the use of fiberoptic bundles with pre-etched microwells at distal ends to assemble dye loaded microspheres. The surface of each spectrally addressed microsphere was attached with a unique bioactive agent and thousands of microspheres carrying different bioaffinity tags combined to form “microspheres array” on pre-etched microwells of fiber optical bundles. More recently, a novel optically encoded microsphere approach was accomplished by using different sized zinc sulfide-capped cadmium selenide nanocrystals incorporated into microspheres. See Nature Biotech. 19, 631-635, (2001). Given the narrow band width demonstrated by these nanocrystals, this approach significantly expands the spectral bar coding capacity in microspheres.
Even though the “spectrally addressed microsphere” approach does provide an advantage in terms of its simplicity over the old fashioned “spatially addressable” approach in microarray making, there remains a need in the art to make the manufacture of biological microarrays less difficult and less expensive and to provide nucleic acid identification systems that are accurate, less complex and less expensive.
One particular problem associated with “spectrally addressed microspheres” lies in the fact that colored compounds typically used in the microspheres are often fluorescent, and hence will provide excessive “background noise” when fluorimetric determinations are performed on the microarray. Another problem inherent in the use of both colorants and latent colorants in polymer microspheres is their propensity to crystallize at the surface of the microspheres or completely or partially wash out of the microspheres. This can provide a hue shift and thus can be a major source of error for this technique. The spatial accessibility of the microsphere's bioaffinity tags to analytes is another area where improvements are needed. Advances in this area can lead to enhanced loading of the tags onto the microspheres and hence a more sensitive array. In addition to the requirements listed, polymer microspheres, which are to be resolved using optical methods, must be relatively monodisperse and must have a diameter of 0.5-50 microns. There are few preparative methods, which can produce polymer microspheres that can fulfill all of these criteria simultaneously.
Macromolecular Rapid Communications Vol. 15 p. 909-915 (1994) reports the immobilization of enzymes to soluble stabilizing polymer arms protruding from the surface of a polymer particle. Enhancements in accessibility of the enzyme to target substrates is observed over enzymes covalently bound directly to the particle/microsphere surface. In this study, however, the enzyme was reversibly adsorbed to the stabilizer arms and was not covalently bound. Furthermore, these polymer microspheres do not contain latent colorants.
JP 2000 178309 discloses highly monodisperse 5-200 micron microspheres with biological macromolecules attached to the surface. The microspheres additionally contain dyes. The dyes described are colorants, not latent colorants and hence will create difficulties when used in bioarrays due to their fluorescence. Furthermore, the method of preparation is laborious.
U.S. Pat. No. 4,837,168 discloses latex particles with biological macromolecules bound to the surface. These particles contain photographic couplers covalently incorporated within the particle structure. These particles, however, are smaller than 100 nanometers. The method of preparation is emulsion polymerization, which will not yield 0.5-20 micron monodisperse particles. Furthermore, these particles are not stabilized by soluble polymers grafted to the surface, but by low molecular weight surfactants.
Many patents exist which disclose latex particles containing photographic couplers. A few representative examples are U.S. Pat. Nos. 3,767, 412, 4,444,870, 6,203,973 and 4,080,211. The disclosed particles are used for silver halide systems, and small particles sizes are necessary. These patents employ preparative methods, such as emulsion polymerization, which yield much smaller particles. Furthermore, the particles described are stabilized by surfactants and not soluble polymers.
In order to use optical imaging to read the test results, the microspheres used should most desirably be 2-20 microns and relatively monodisperse, in addition to containing the latent colorant. There are few methods, which may produce microspheres, which meet all of these specifications.